Method of coupling binding agents to a substrate surface

ABSTRACT

The present invention relates to a method of coupling multiple binding agents to respective areas of a substrate surface by hydrodynamic addressing, using two laminar fluid flows that flow together in the same direction over the substrate surface with an interface to each other to successively couple the binding agents to the substrate areas, wherein each successive coupling of a binding agent to a surface area is followed or preceded by selective deactivation or activation of a selected surface area according to a defined protocol. The invention also relates to the use of such a binding agent-coupled substrate surface for analytical purposes.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/384,626 filed May 31, 2002, and Swedish PatentApplication No. 0201637-6 filed May 31, 2002, both of which applicationsare incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method of coupling bindingagents to a substrate surface by passing a binding agent-containingfluid flow over the surface, and more particularly by using hydrodynamicaddressing techniques to selectively direct fluid flows to desiredsurface areas. The invention also relates to analytical use of themethod.

[0004] 2. Description of the Related Art

[0005] Flow cells are used extensively nowadays in a variety ofanalytical systems. Typically, the flow cell has an inlet opening, aflow channel with a sensing surface, and an outlet opening. A samplefluid to be investigated is introduced through the inlet opening, passesthrough the flow channel and leaves the flow cell through the outletopening. The flow cell may have more than one inlet opening andoptionally more than one outlet opening to permit desired manipulationsof the flow pattern within the flow cell.

[0006] The sensing surface usually comprises a substance layer to whicha recognition element for an analyte in the sample is immobilised,typically a biochemical affinity partner to the analyte. When theanalyte interacts with the recognition element, a physical or chemicalchange is produced on the sensing surface that can be detected by adetector, e.g., an optical, electrochemical or calorimetric detector. Aflow channel may contain two or more sensing surfaces with differentrecognition elements.

[0007] The sensing surface or surfaces in the flow cell may befunctionalized, or sensitized, in situ, i.e., within the flow cell. WO90/05305 discloses a method for functionalising a sensing surface havingfunctional groups thereon by passing a reagent solution containing a bi-or polyfunctional ligand over the surface, the ligand having a functionwhich immobilizes the ligand on the sensing surface and at least onemore function which is exposed on the sensing surface for interactionwith the analyte. In a specific embodiment, the sensing surface has abound carboxymethyl-dextran layer. After activation of the surfacethrough derivatization with N-hydroxysuccinimide, mediated byN-ethyl-N′-(dimethylaminopropyl)carbodiimide, a ligand in the form ofaminotheophylline or aminobiotin is coupled to the activated surface.

[0008] WO 99/36766 discloses methods and systems using hydrodynamicaddressing techniques to allow immobilization of different ligands todiscrete sensing areas within a single flow cell channel, as well as topermit controlled sample delivery to such sensitized areas. For a Y-typeflow cell, which has an inlet end with two inlet ports and an outlet endwith one outlet port, and a sensing surface between the ends, WO99/36766 describes sensitization of two spaced apart sensing areas withdifferent ligands. This is done by providing a laminar flow of asensitising fluid adjacent to a laminar flow of non-sensitizing(blocking) fluid such that the fluids flow together over the sensingsurface with an interface to each other. By adjustment of the relativeflow rates of the two fluids the interface may be positioned laterallysuch that sensitising fluid selectively contacts a desired area of thesensing surface. More specifically, if the sensitizing fluid initiallycontains a first ligand capable of binding to the sensing surface andthe interface is positioned such that the sensitizing fluid covers, say,about one third of the lateral extension of the sensing surface, and theblocking fluid covers the remaining two thirds, the first ligand will beimmobilized to the first-mentioned third of the sensing surface. Then,the sensitizing fluid is replaced by blocking fluid, and the blockingfluid is replaced by a sensitizing fluid containing a second ligand. Bypositioning the interface such that the sensitizing fluid again coversabout one third of the lateral extension of the sensing surface, now,however, at the opposite side of the flow path, the second ligand willbe immobilized to that area, thereby providing a sensing surface which,as seen laterally, has about one third immobilized with the firstligand, about one third immobilized with the second ligand, and anintermediate non-immobilized third which only has been in contact withblocking fluid and may suitably be used as a reference area.

[0009] For providing more than two differently sensitized sensingsurface areas, WO 99/36766 discloses the use of a so-called in-type flowcell having three inlets and a single outlet. In this embodiment, alaminar flow of sensitizing fluid is sandwiched between two laminarflows of blocking fluid, and the sensitizing fluid may thereby bedisplaced laterally to selectively contact a number of sensing surfaceareas.

[0010] A similar use of a ψ-type flow cell is disclosed in WO 00/56444.

[0011] While the ψ-type flow cell is advantageous in comparison with theY-type flow cell in that the former readily permits sensitization withmore than two different ligands, the ψ-type flow cell requires the useof an additional pump (one pump for each fluid flow inlet), whichconsiderably complicates the control of the different laminar flows. Itwould therefore be desired to be able to use a Y-type flow cell tosensitize a sensing surface with more than two different ligands.

[0012] Accordingly, it is an object of the present invention to providea method which permits coupling of multiple different ligands, orgenerally binding agents, to respective surface areas by hydrodynamicaddressing using only two adjacent laminar fluid flows, such as e.g., ina Y-type flow cell.

BRIEF SUMMARY OF THE INVENTION

[0013] The above and other objects and advantages are obtained by anovel method of coupling multiple binding agents to respective areas ofa substrate surface by hydrodynamic addressing, wherein each successivecoupling of a binding agent to a substrate area is followed or precededby selective deactivation or activation of a selected surface area orareas according to a particular coupling protocol.

[0014] In one aspect, the present invention provides a method ofcoupling at least two different binding agents to respective definedareas of a substrate surface by hydrodynamic addressing based on twolaminar fluid flows that flow together in the same direction over thesubstrate surface with an interface to each other, and selectivelycontacting a defined area of the substrate surface with a desired fluidby positioning the interface laterally through adjustment of therelative flow rates of the two fluid flows, which method comprises atleast one of hydrodynamic addressing procedures A and B, whereinprocedure A comprises immobilizing a first binding agent to a first areaof the substrate surface by contacting the area with a fluid containingthe first binding agent, deactivating the first area by subjecting thearea to a deactivating fluid, and immobilizing a second binding agent toa second area of the substrate surface by contacting a substrate surfacearea including the first and second areas with the second binding agent;and procedure B comprises deactivating a first area of the substratesurface by subjecting the area to a deactivating fluid, immobilizing afirst binding agent to a second area of the substrate surface bycontacting the first and second areas with a fluid containing the firstbinding agent, activating at least a part of the first area bysubjecting the area to an activating fluid, and immobilizing a secondbinding agent to the first area by contacting the first area with afluid containing the second binding agent.

[0015] In one embodiment, the method comprises the steps of:

[0016] a) providing a substrate surface, at least a part of which isreactive (e.g., activated) to permit coupling of binding agents thereto;

[0017] b) passing over the substrate surface a laminar flow of a fluidcontaining a first binding agent, and adjacent thereto a laminar flow ofa blocking fluid that does not interact with the substrate surface suchthat the two fluids flow together in the same direction with aninterface to each other, and adjusting the relative flow rates of thetwo laminar fluid flows to position the interface such that the firstbinding agent-containing fluid selectively contacts a first reactive(e.g., activated) area of the substrate surface to couple the firstbinding agent thereto;

[0018] c) replacing the flow of the first binding agent-containing fluidwith a laminar flow of a deactivating fluid, and adjusting the relativeflow rates of the two laminar fluid flows to position or displace theinterface laterally such that the deactivating fluid selectivelycontacts at least the first binding agent-coupled area but less than thewhole activated surface area for deactivation thereof;

[0019] d) replacing the flow of the deactivating fluid with a laminarflow of a fluid containing a second binding agent, and adjusting therelative flow rates of the two laminar fluid flows to displace theinterface laterally such that the second binding agent-containing fluidselectively contacts the deactivated area and an adjacent secondreactive (e.g., activated) area of the substrate surface to selectivelycouple the second binding agent to the second area; and, optionally,

[0020] e) replacing the flow of the second binding agent-containingfluid with a laminar flow of deactivating fluid, and adjusting therelative flow rates of the two laminar flows to position or displace theinterface laterally such that the deactivating fluid selectivelycontacts at least the deactivated area and the second bindingagent-coupled area of the substrate surface for deactivation thereof.

[0021] Another embodiment of the method comprises the steps of:

[0022] a) providing a substrate surface, at least part of which isreactive to permit coupling of binding agents thereto;

[0023] b) passing over the substrate surface a laminar flow of adeactivating fluid, and adjacent thereto a laminar flow of a blockingfluid that does not interact with the substrate surface, such that thetwo fluids flow together in the same direction with an interface to eachother, and adjusting the relative flow rates of the two laminar fluidflows to position the interface such that the deactivating fluidselectively contacts a first reactive area of the substrate surface fordeactivation thereof;

[0024] c) replacing the flow of the deactivating fluid with a laminarflow of a fluid containing a first binding agent, and adjusting therelative flow rates of the two laminar fluid flows to displace theinterface laterally such that the first binding agent-containing fluidselectively contacts the deactivated area and an adjacent secondreactive area of the substrate surface to selectively couple the firstbinding agent to the second area;

[0025] d) replacing the flow of the first binding agent-containing fluidwith a laminar flow of activating fluid, and adjusting the relative flowrates of the two laminar fluid flows to displace the interface laterallysuch that the activating fluid selectively contacts at least a part ofthe deactivated first area for activation thereof; and

[0026] e) replacing the flow of the activating fluid with a laminar flowof a fluid containing a second binding agent, and adjusting the relativeflow rates of the two laminar fluid flows to position or displace theinterface laterally such that the second binding agent-containing fluidselectively contacts the activated first area to selectively couple thesecond binding agent thereto.

[0027] Optionally, combinations of the above two method embodiments mayalso be used.

[0028] In another aspect, the present invention provides the use of themethod for analysing a fluid sample for the presence of at least oneanalyte.

[0029] In still another aspect, the present invention provides the useof the method for studying interactions of at least one analyte with thesubstrate surface.

[0030] In yet another aspect, the present invention provides a computerprogram product comprising program code means stored on a computerreadable medium or carried on an electrical or optical signal forperforming the method.

[0031] These and other aspects of this invention will be evident uponreference to the attached drawings and the following detaileddescription.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0032]FIG. 1 is a schematic illustration of an embodiment of the methodof the present invention using a Y-type flow cell.

[0033]FIG. 2 is a schematic illustration of the different steps in themethod relating to FIG. 1.

[0034]FIG. 3 is a schematic illustration of another method embodiment ofthe present invention using a Y-type flow cell.

DETAILED DESCRIPTION OF THE INVENTION

[0035] As mentioned above, the present invention is generally directedto the coupling of multiple binding agents to a substrate (solidsupport) surface using hydrodynamic addressing techniques tosuccessively bring binding agent-containing fluid flows into selectivecontact with different surface areas to couple the different bindingagents thereto. More particularly, two adjacent laminar fluid flows areprovided which flow together over the substrate surface with aninterface between them, as described in the aforementioned WO 99/36766(the entire disclosure of which is incorporated by reference herein).

[0036] By adjusting the relative flow rates of the two fluid flows, theinterface may be positioned laterally to make the different fluidscontact respective desired areas of the substrate surface. To couple abinding agent to the substrate surface, one of the fluid flows containsa binding agent, and the other fluid flow is one that can not interactwith the substrate surface, below frequently referred to as a blockingfluid. To permit coupling of the binding agent to the substrate surface,the surface should, of course, be sufficiently reactive towards thebinding agent. Preferably, the surface is activated by an activatingagent as is per se well known in the art.

[0037] According to the present invention, a binding agent may becoupled to a (preferably activated) surface followed by selectivedeactivation of the coupled surface area, and optionally of an adjacentarea but less than the whole activated surface, and a different bindingagent is then coupled to an adjacent activated surface area, followed bydeactivation of the activated surface area. By proceeding in this mannerwith successive coupling of binding agents with deactivation after eachcoupling, multiple binding agents may readily be coupled to respectiveareas of an activated substrate surface, if desired with non-coupledareas between the coupled areas.

[0038] Optionally, the procedure may start with deactivating an edgearea of the activated substrate surface, and a binding agent is thencoupled to an activated area adjacent to the deactivated edge area.

[0039] While it is possible to carry out the coupling procedure of theinvention from one side of the flow path to the other, it may beconvenient to start from one side and successively couple a first numberof binding agents, and then shift to the opposite side of the flow pathand successively couple a second number of binding agents from thatside. In this case, activation of the surface area to be coupled withthis second number of agents may take place after coupling the firstnumber of binding agents.

[0040] Alternatively, a binding agent may be coupled to a (preferablyactivated) surface by selective deactivation of a part of the activatedsubstrate surface, coupling of a binding agent to the remainingactivated area, reactivation of at least a part of the deactivated area,and coupling of a different binding agent to the reactivated area. Byproceeding in this way with successive activation and deactivationbefore each coupling of binding agent, multiple binding agents may beimmobilized to respective areas of an activated surface.

[0041] Optionally, a pre-activated surface may be used instead ofactivating the surface areas at the time of coupling the binding agents.

[0042] It is not necessary that all the binding agents coupled to thesubstrate surface be different. However, usually at least two adjacentbinding agent coupled areas should not support the same binding agent.

[0043] Preferably, at least one surface area is not coupled with bindingagent and used as a reference area(s).

[0044] The term “binding agent” as used herein means any agent that is amember of a specific binding pair, including, for instance polypeptides,such as, e.g., proteins or fragments thereof, including antibodies;nucleic acids, e.g., oligonucleotides, polynucleotides, and the like;etc. The binding agent is often a ligand.

[0045] The term “ligand” as used herein means a molecule that has aknown or unknown affinity for a given analyte and can be immobilized ona predefined region of the surface. The ligand may be a naturallyoccurring molecule or one that has been synthesized. The ligand may beused per se or as aggregates with another species. Optionally, theligand may also be a cell.

[0046] The term “reactive” with respect to a substrate surface meansthat the surface should exhibit a binding moiety, such as e.g., afunctional group, capable of coupling to a binding agent.

[0047] The term “activation” means modification of a substrate surfaceto enable coupling a binding agent thereto, usually modification of afunctional group on the substrate surface.

[0048] The term “deactivation” means modification of a reactivesubstrate surface, usually of an activated functional group thereon,such that coupling of a binding agent to the surface is substantiallyprevented. Deactivation may include restoring an original functionalgroup or making a reactive functional group or other reactive moietyinactive in other ways.

[0049] Activating and deactivating agents that may be used for thepurposes of the present invention are per se well known to a personskilled in the art and may readily be selected for each particularsituation.

[0050] The choice of activating agent (and method) depends, of course,on the functional group to be activated and on the desired reactivegroup to be obtained by the activation, which in turn depends on thebinding agent to be coupled to the substrate surface. Exemplaryfunctional group/activating agent combinations include those introducinghydroxysuccinimide esters, nitro- and dinitrophenyl esters, tosylates,mesylates, triflates and disulfides. For example, a hydroxy group may bereacted to activated ester with disuccinic carbonate, or to epoxide witha diepoxide. A carboxy group may be activated to N-hydroxysuccinimideester by reaction with N-hydroxysuccinimide (NHS) and carbodiimide,e.g., 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide (EDC), or todinitrophenyl ester by reaction with dinitrophenol. A thiol (mercapto)group may be activated to a disulfide group by reaction with e.g., adipyridyldisulfide or (2-pyridinyldithio)ethaneamine.

[0051] For example, NHS/EDC activation of carboxy groups may be used tocouple binding agents having an amine function (so-called aminecoupling) or an aldehyde function (so-called aldehyde coupling).

[0052] NHS/EDC activation may also be used to introduce thiol groups,e.g., by reaction with dithioerythritol (DTE). These thiol groups maythen either (i) be reacted with an active disulfide group of a bindingagent (so-called surface thiol coupling), or (ii) be activated todisulfide groups which may be reacted with a thiol function of a bindingagent (so-called ligand thiol coupling).

[0053] Also avidin or streptavidin may be coupled to anNHS/EDC-activated surface to permit capture of a biotinylated bindingagent (so-called avidin coupling).

[0054] The choice of deactivating agent depends, of course, on theactive group(s) to be deactivated. For example, N-hydroxysuccinimideesters may be deactivated with ethanolamine or sodium hydroxide,deactivation with sodium hydroxide being reversible, i.e., thedeactivated surface may be reactivated by activation with an activatingagent.

[0055] The term “coupling” as used herein is to be interpreted in abroad sense and includes covalent binding as well as other types ofbinding.

[0056] The method of the invention is preferably performed in a flowcell. Suitable flow cells for use in the present invention may assume anumber of forms, the design of which may vary widely. A preferred typeof flow cell is the “Y-flow cell” which has two inlets and one outletand which is described in, for example, the above-mentioned WO 99/36766.

[0057] The substrate surface is usually a sensing surface, which term inthe present context is to be construed broadly. The sensing surface may,for example, be a surface or surface layer that can interactspecifically with a species present in a fluid, a surface or surfacelayer that can be chemically or physically sensitised to permit suchinteraction, or a surface or surface layer that can be chemically orphysically activated to permit sensitisation thereof. A flow cell maycontain one or more sensing surfaces.

[0058] Binding events at the sensing surface may be detected by numeroustechniques. In many cases it is favourable to use so-called non-labelmethods. Representative such detection methods include, but are notlimited to, mass detection methods, such as piezoelectric, optical,thermo-optical and surface acoustic wave (SAW) device methods, andelectrochemical methods, such as potentiometric, conductometric,amperometric and capacitance/impedance methods. With regard to opticaldetection methods, representative methods include those that detect masssurface concentration, such as reflection-optical methods, includingboth external and internal reflection methods, angle, wavelength,polarization, or phase resolved, for example evanescent waveellipsometry and evanescent wave spectroscopy (EWS, or internalreflection spectroscopy), both may include evanescent field enhancementvia surface plasmon resonance (SPR), Brewster angle refractometry,critical angle refractometry, frustrated total reflection (FTR),scattered total internal reflection (STIR), which may include scatterenhancing labels, optical wave guide sensors; external reflectionimaging, evanescent wave-based imaging such as critical angle resolvedimaging, Brewster angle resolved imaging, SPR-angle resolved imaging,and the like. Further, photometric and imaging/microscopy methods, “perse” or combined with reflection methods, based on for example surfaceenhanced Raman spectroscopy (SERS), surface enhanced resonance Ramanspectroscopy (SERRS), evanescent wave fluorescence (TIRF) andphosphorescence may be mentioned, as well as waveguide interferometers,waveguide leaking mode spectroscopy, reflective interferencespectroscopy (RIfS), transmission interferometry, holographicspectroscopy, and atomic force microscopy (AFR).

[0059] SPR spectroscopy may be mentioned as an exemplary commerciallyavailable analytical system to which the present invention may beapplied. One type of SPR-based biosensors is sold by Biacore AB(Uppsala, Sweden) under the trade name BIACORE®. These biosensorsutilize a SPR based mass-sensing technique to provide a “real-time”binding interaction analysis between a surface bound ligand and ananalyte of interest.

[0060] The basic principles of the invention will now be furtherdescribed with reference to FIG. 1. A Y-type flow cell, generallydesignated by reference numeral 1, has two inlets 2 and 3, respectively,and an outlet 4. The flow cell has a sensing surface 5 on a wall portionthereof. The sensing surface may, for example, be of the type describedin U.S. Pat. Nos. 5,242,828 and 5,436,161 (the full disclosures of whichare incorporated by reference herein) and may, for instance, include amatrix coating in the form of carboxymethylated dextran.

[0061] A laminar flow of a first fluid, indicated by arrow 6, isintroduced through inlet 2, and a laminar flow of a second fluid,indicated by arrow 7, is introduced through inlet 3 such that the twofluids flow together over the sensing surface 5 with an interface (notshown) between them, exiting through outlet 4, as indicated by arrow 8.By adjusting the relative flow rates of the two fluid flows, theposition of the interface may be displaced laterally as desired and beset at any distance from either side wall of the flow cell.Immobilization of three different ligands to the sensing surface 5 isperformed as described in steps (1) to (10) below. Reference issimultaneously made to FIG. 2, which schematically illustrates theprocedure. Each square in FIG. 2 represents a Y-cell, as shown in FIG.1, with the respective process step number indicated at the top thereof.

[0062] (1) The procedure is started with the first fluid 6 being a fluidcontaining an activating agent, and the second fluid 7 being a blockingfluid (i.e., one that does not affect or interact with the sensingsurface), such as buffer. The interface between the two fluid flows ispositioned such that the activating fluid selectively covers a sensingsurface area which, in the illustrated case, extends over more than halfthe lateral extension of the sensing surface as indicated by arrow 9. Ifthe sensing surface 5 includes a layer of carboxymethylated dextran assuggested above, the activating agent may be N-hydroxysuccinimide (NHS)together with N-ethyl-N′-(dimethylaminopropyl)-carbodiimide (EDC). Itis, alternatively, possible to activate the whole sensing surface byreplacing the two fluid flows by a single flow of activating fluid.

[0063] (2) The activating fluid is then replaced by a fluid containing adeactivating agent, and the interface between the two fluid flows 6, 7is positioned at the point of arrow 10, such that the deactivating fluidselectively covers an area 11 close to the flow cell wall 12, the bufferfluid covering the rest of the sensing surface 5. The area 11 willthereby be deactivated so as not to react with ligand-containing fluidin the following step. If NHS/EDC is used as activating agent, thedeactivating agent may, for example, be ethanolamine.

[0064] (3) The deactivating fluid 6 is then replaced by a fluidcontaining a first ligand, e.g., a first monoclonal antibody, and theinterface between the two fluid flows 6, 7 is positioned at the point ofarrow 13, such that the ligand-containing fluid selectively covers thedeactivated area 11 as well as an adjacent activated area 14. The area14 will thereby have the first ligand coupled thereto.

[0065] (4) The ligand-containing fluid 6 is then replaced bydeactivating fluid, and the interface between the two fluid flows 6, 7is positioned at the point of arrow 15, such that the deactivating fluidselectively covers areas 11, 14 and an adjacent activated area 16 whichwill thereby be deactivated.

[0066] (5) The deactivating fluid 6 is then replaced by a fluidcontaining a second ligand, e.g., a second monoclonal antibody, and theinterface between the two fluid flows 6, 7 is positioned at the point ofarrow 9, such that the ligand-containing fluid covers areas 11, 14, 16and an adjacent activated area 17 extending between the points of arrows9 and 15. The area 17 will thereby have the second ligand coupledthereto.

[0067] (6) The ligand-containing fluid 6 is then replaced by adeactivating fluid, and the interface between the two fluid flows 6, 7is positioned at the point of arrow 9, such that the deactivating fluidcovers areas 11, 14, 16 and 17 which will thereby be deactivated.

[0068] (7) The ligand-containing fluid 6, introduced through inlet 2, isthen replaced by buffer, and the buffer flow 7, introduced through inlet3, is replaced by activating fluid. The interface between the two fluidflows 6, 7 is positioned such that the activating fluid selectivelycovers an area extending as indicated by arrow 18, i.e., from the flowcell wall 19 towards but not up to the ligand-coupled area 17.

[0069] (8) The activating fluid 7 is then replaced by deactivatingfluid, and the interface between the two fluid flows 6, 7 is positionedat the point of arrow 20 such that the deactivating fluid selectivelycovers an activated area 21 close to the flow cell wall 19 which area isthereby deactivated.

[0070] (9) The deactivating fluid 7 is then replaced by a fluidcontaining a third ligand, e.g., a third monoclonal antibody, and theinterface between the two fluid flows 6, 7 is positioned at the point ofarrow 19, such that the ligand-containing fluid covers the area 21 andan adjacent activated area 22 extending between the points of arrows 18and 20. This area 22 will thereby have the third ligand coupled thereto.

[0071] (10) Finally, the ligand-containing fluid 7 is replaced bydeactivating fluid, and the interface between the two fluid flows 6, 7is positioned at the point of arrow 9 such that the deactivating fluid 7selectively covers the deactivated area 21, the ligand-coupled area 22and (in the illustrated case) a non-activated area 23 between theligand-coupled areas 22 and 17 for deactivation thereof. (Alternatively,the deactivating fluid may only cover the areas 21 and 22).

[0072] The sensing surface 5 now exhibits three discrete sensing areas14, 17 and 22, each supporting a different ligand, which sensing areasare separated mutually as well as to the flow cell walls 12, 19 bydeactivated areas 11, 16, 21 and 23. The resulting Y-cell with thedesired sensitized sensing surface is also shown in FIG. 2, to the rightof the step 10 Y-cell.

[0073] While in the above illustrated case three discrete areas havebeen immobilised with ligand, it is understood that by proceeding asdescribed above with successive ligand-coupling and deactivation steps,from e.g., four, five or six to considerably more ligand-coupled areasmay likewise readily be produced on a sensing surface, depending onamong other things the flow cell, the size of the sensing surface, theprecision and control of pumps, etc.

[0074] In the above described immobilisation procedure, the differentsensing surface areas are immobilised from the edges of the surfacetowards the centre. This requires, however, that ligands have to becontacted with areas to which a ligand has already been immobilised.This may be avoided by a procedure in which the ligands are immobilisedto the respective areas from the centre towards the edges. An embodimentof such a procedure is outlined below with reference to FIG. 3.

[0075] In FIG. 3, the flow cell 30 is a Y-cell similar to that shown inFIGS. 1 and 2, having two inlets 31, 32, an outlet 33 and a sensingsurface on a wall portion thereof (not specifically indicated). Threedetection spots, i.e., areas on the sensing surface that are measured bya detection system (not shown) are indicated at 34, 35 and 36. A numberof images of flow cell 30, numbered from 1 to 9, are shown to illustratethe different steps of the procedure. The sensing surface may e.g., becarboxymethylated dextran, the activating fluid may be anNHD/EDC-solution, deactivating fluids may be sodium hydroxide solutionand ethanolamine solution, and the blocking fluid buffer.

[0076] (1) The procedure is started by passing a laminar flow ofactivating fluid through inlet 31 and blocking fluid through inlet 32.In the illustrated case, the interface between the two fluid flows ispositioned such that the activating fluid covers detection spots 34 and35 (i.e., two thirds of the lateral extension of the sensing surface).

[0077] (2) The activating fluid is then replaced by deactivating fluid,and the interface is positioned such that the deactivating fluid coversdetection spot 34 (i.e., the left third of the sensing surface) todeactivate that part of the activated surface area, leaving the centrearea that includes detection spot 35 activated. The deactivating fluidis selected such that the deactivated area may later be reactivated(e.g., sodium hydroxide as mentioned above).

[0078] (3) The deactivating fluid is then replaced by a fluid containinga first ligand, and the interface is positioned such that theligand-containing fluid covers detection spots 34 and 35, i.e., the samearea as that activated in step (1) above. Due to the deactivating step(2), the first ligand will only couple to the still activated centrearea including detection spot 35.

[0079] (4) The ligand-containing fluid is then replaced by activatingfluid, and the interface is positioned such that the activating fluidcovers the area that was deactivated in step (2) above (i.e., the leftthird of the sensing surface in FIG. 3).

[0080] (5) The activating fluid is then replaced by a fluid containing asecond ligand, and the interface is positioned such thatligand-containing fluid covers the area that was activated in step (4)above. Thereby, the second ligand will be coupled to this area. Thesensing surface now has the second ligand coupled to the left area thatincludes detection spot 34, and the first ligand to the centre area thatincludes detection spot 35.

[0081] (6) The ligand-containing fluid supplied through inlet 31 is thenreplaced by blocking fluid, and the blocking fluid supplied throughinlet 32 is replaced by activating fluid. The interface is nowpositioned such that the activating fluid covers the right third of thesensing surface to activate that area.

[0082] (7) The activating fluid is then replaced by a fluid containing athird ligand, and the interface is maintained in the same position as instep (6) above to thereby couple the third ligand to the area that wasactivated in step (4) and includes detection spot 36.

[0083] (8) The activating fluid through inlet 31 is then replaced bydeactivating fluid and inlet 32 is closed to deactivate all threeligand-coupled areas of the sensing surface. In this case thedeactivating fluid need not be one that permits reactivation as in step(2) and must not affect the immobilised ligands, e.g., ethanolamine.

[0084] (9) The sensing surface now has three different ligands coupledthereto, i.e., the second ligand coupled to a lateral area includingdetection spot 34, the first ligand coupled a central area includingdetection spot 35, and the third ligand coupled to a lateral areaincluding detection spot 36.

[0085] It is readily seen that ligands may readily be coupled to morethan three different areas by following the above-described procedure.

[0086] An alternative immobilization procedure, which partly avoidscontacting a ligand with an area or areas to which ligands have alreadybeen immobilized, and does not require reactivation of a deactivatedsurface, will be schematically described below with reference to FIG. 3again.

[0087] (1) Half the sensing surface is first activated by introducingactivating fluid through inlet 31 and blocking fluid through inlet 32.

[0088] (2) A first ligand is then immobilized to an inner part (i.e.,adjacent to the centre) of the activated area by introducing ligandthrough inlet 32 and blocking fluid through inlet 31, and positioningthe fluid interface in the middle of the activated area.

[0089] (3) A second ligand is then immobilized to the remaining part ofthe activated area (i.e., adjacent to the flow cell wall) by introducingligand through inlet 31 and blocking fluid through inlet 32.

[0090] (4) After deactivation of the areas coupled with the first andsecond ligands, respectively, third and fourth ligands may then beimmobilized to the other half of the sensing surface by activating thisarea and successively coupling the ligands, introduced through inlet 31,according to the method variant described above with reference to FIGS.1 and 2, i.e., with intermediate deactivation of the inner area coupledwith the third ligand before introducing the fourth ligand.

[0091] Alternatively, a third ligand may be coupled to the remainingpart of the activated area by introducing the ligand through inlet 32.

[0092] By proceeding as described above with reference to FIG. 3, it isalso possible to couple a fourth ligand without the ligand-containingfluid having to pass areas with immobilized ligands, i.e., bydeactivating an outer part of the activated area (adjacent to the flowcell wall) prior to the introduction of the third ligand through inlet32, reactivating the deactivated area after coupling of the thirdligand, and subsequently introducing the fourth ligand through inlet 32to couple the ligand to the reactivated area.

[0093] Numerous applications of a sensing surface to which multipleligands have been immobilised at discrete areas as described above arereadily apparent to a person skilled in the art and need not be detailedherein.

[0094] The invention will now be illustrated further by the followingnon-limiting examples.

EXAMPLE 1

[0095] Immobilization of Three Different Ligands in a Y-Cell

[0096] A BIACORE® S51 instrument (Biacore AB, Uppsala, Sweden) was used.This instrument has two Y-type flow cells which allow a dual flow offluids over a sensor chip surface, so-called hydrodynamic addressing, asdescribed in WO 99/36766 mentioned above. The instrument uses threeparallel detection spots on the sensor chip, each detection spotoccupying one diode row in a diode array detector for detecting lightreflected at the detection spots on the sensor chip surface. Thedetection spots are mutually spaced by one diode row. As sensor chip wasused Sensor Chip CM5 (Biacore AB, Uppsala, Sweden) which has agold-coated surface with a covalently linked carboxymethyl-modifieddextran polymer hydrogel. Running buffer was HBS-N (10 mM HEPES pH 7.4and 150 mM NaCl) (Biacore AB, Uppsala, Sweden). The output from theinstrument is a “sensorgram” which is a plot of detector response(measured in “resonance units”, RU) as a function of time. An increaseof 1000 RU corresponds to an increase of mass on the sensor surface ofapproximately 1 ng/mm².

[0097] Antibodies anti-IL-8, anti-IL-10 and anti-IL-12 againstinterleukin 8 (IL-8), interleukin 10 (IL-10) and interleukin 12 (IL-12)(in-house sources, Biacore AB, Uppsala, Sweden), were diluted 10 timesfrom stock solution with 10 mM acetate pH 4.5. They were thenimmobilized in the two flow cells of the instrument by the hydrodynamicaddressing procedure described above with reference to FIGS. 1 and 2,using 10 minutes activation with EDC/NHS and sequential deactivation andligand (antibody) immobilization. Each deactivation was performed withethanolamine (100 mM, pH 8.5) for two minutes, and each antibody wasinjected for 7 minutes.

[0098] The different antibodies were immobilized in parallel stripesextending through the spots used for detection in the BIACORE® S51instrument, meaning that with regard to the detector there was one dioderow between each immobilized spot. However, in order to ensure that theimmobilized antibodies within the detection spots were homogenous, eachantibody was immobilized such that it extended into one third of theadjacent interspaces. The resulting immobilization levels in resonanceunits (RU) for the different ligands are shown in Tables 1 (flow cell 1)and 2 (flow cell 2) below. Bold values show targeted immobilizationspots. Indicated in italics below each immobilization level value is thepercentage immobilized, obtained by subtracting the baseline beforeinjection of antibody from the baseline after subsequent deactivationwith ethanolamine. TABLE 1 Immobilized Diode row number (flow cell 1)antibody 1 2 3 4 5 anti-IL-8 31480 18821 280 189 116 100.0 46.0 −0.3−0.5 −0.1 anti-IL-10 31917 28574 26629 18645 62 −14.4 32.2 100.0 47.7−0.1 anti-IL-12 23892 14945 13037 9904 15611 −6.1 −20.3 −13.2 21.2 100.0

[0099] TABLE 2 Immobilized Diode row number (flow cell 2) antibody 1 2 34 5 anti-IL-8 123 152 147 20214 30138 −0.1 −0.3 −0.2 49.6 100.0anti-IL-10 98 17142 26016 27174 30496 0.1 49.3 100.0 20.6 −13.0anti-IL-12 16013 9407 13525 13877 23681 100.0 14.5 −8.7 −18.8 −12.0

[0100] As shown in the tables, selective repeatable immobilization ofligands at the desired detection spots was obtained with no or very low“cross-talk” between spots.

[0101] The corresponding antigens IL-8, IL-10 and IL-12 were diluted to500 ng/ml in HBS-N (Biacore AB, Uppsala, Sweden) and sequentiallyinjected for 4 minutes in both flow cells. Between injections, thesurfaces were regenerated with 0.1 trifluoroacetic acid (TFA) for 6seconds. The resulting binding levels (in RU) are shown in Tables 3 and4 below. Below each binding value, calculated percent cross-talk isindicated in italics. TABLE 3 Analyte Diode row number (flow cell 1)antigen 1 2 3 4 5 IL-8 1611 776 25 14 2 100.0 48.2 1.5 0.9 0.1 IL-10 2287 484 6 −1 0.5 59.4 100.0 1.3 −0.3 IL-12 1 4 8 450 1076 0.1 0.4 0.841.8 100.0

[0102] TABLE 4 Analyte Diode row number (flow cell 2) antigen 1 2 3 4 5IL-8 4 16 26 730 1451 0.3 1.1 1.8 50.3 100.0 IL-10 −3 8 328 272 1 −0.92.5 100.0 83.1 0.3 IL-12 1183 396 7 2 −2 100.0 33.4 0.6 0.2 −0.2

[0103] As shown in the tables above, cross-talk between spots was below1%, except for IL-8 which bound by 1.5% and 1.8%, respectively, to theanti-IL-10 antibody, most likely due to cross-reactivity.

EXAMPLE 2

[0104] Immobilization of Five Different Ligands in a Y-Cell

[0105] Following the procedure in Example 1 and using the sameinstrument and sensor chip, five different ligands were immobilized in aY-cell. In this case, however, each ligand spot adjoined to the next onewithout any separating detector diode row. The interfaces between thespots were adjusted to obtain a 5% theoretical cross-talk to ensurehomogeneity of the spots. The following ligands were sequentiallyimmobilized: anti-IL-8, anti-myoglobin, anti-IL-10, anti-CKMB andanti-IL-12 (all from in-house sources, Biacore AB, Uppsala, Sweden). Theresulting immobilization levels for flow cell 1, as well as thepercentages cross-talk (in italics), are shown in Table 5 below. TABLE 5Immobilized Diode row number (flow cell 2) antibody 1 2 3 4 5 anti-IL-827550 910 253 180 127 3 1 1 0 anti- 32199 29280 1149 182 138 myoglobin 30 0 anti-IL-10 29762 26385 24325 1024 123 4 0 anti-IL-12 22680 1594012330 1763 24848 1 0 0 −2 anti-CKMB 22769 16003 12690 19940 26576 0 0 2

[0106] As appears from the table above, all the antibody immobilizationswere successful, the maximum cross-talk obtained being 4%.

[0107] The antigens corresponding to the immobilized antibodies werethen injected as described in Example 1, except that myoglobin and CKMBwere each diluted to 5 μg/ml in HBS-N. The resulting binding level foreach analyte in percent of that for the “active” spot (after subtractionof the average for buffer samples) are presented in Table 6 below. TABLE6 Analyte Diode row number (flow cell 2) antigen 1 2 3 4 5 myoglobin 2.9100.0 9.3 0.5 0.0 CKMB −2.4 −2.5 0.5 100.0 3.7 IL-8 100.0 3.9 1.8 1.0−0.1 IL-10 −2.6 −2.2 100.0 −0.3 −2.8 IL-12 −0.4 −0.4 1.5 3.1 100.0

[0108] As shown in the table above, the maximum cross-talk was below10%.

[0109] It is to be understood that the invention is not limited to theparticular embodiments of the invention described above, but the scopeof the invention will be established by the appended claims.

1. A method of coupling at least two different binding agents torespective defined areas of a substrate surface by hydrodynamicaddressing based on two laminar fluid flows that flow together in thesame direction over the substrate surface with an interface to eachother, and selectively contacting a defined area of the substratesurface with a desired fluid by positioning the interface laterallythrough adjustment of the relative flow rates of the two fluid flows,which method comprises at least one of hydrodynamic addressingprocedures A and B, wherein: procedure A comprises immobilizing a firstbinding agent to a first area of the substrate surface by contacting thearea with a fluid containing the first binding agent, deactivating thefirst area by subjecting the area to a deactivating fluid, andimmobilizing a second binding agent to a second area of the substratesurface by contacting a substrate surface area including the first andsecond areas with the second binding agent; and procedure B comprisesdeactivating a first area of the substrate surface by subjecting thearea to a deactivating fluid, immobilizing a first binding agent to asecond area of the substrate surface by contacting the first and secondareas with a fluid containing the first binding agent, activating atleast a part of the first area by subjecting the area to an activatingfluid, and immobilizing a second binding agent to the first area bycontacting the area with a fluid containing the second binding agent. 2.The method according to claim 1, wherein procedure A comprises the stepsof: a) providing a substrate surface, at least part of which is reactiveto permit coupling of binding agents thereto; b) passing over thesubstrate surface a laminar flow of a fluid containing a first bindingagent, and adjacent thereto a laminar flow of a blocking fluid that doesnot interact with the substrate surface, such that the two fluids flowtogether in the same direction with an interface to each other, andadjusting the relative flow rates of the two laminar fluid flows toposition the interface such that the first binding agent-containingfluid selectively contacts a first reactive area of the substratesurface to couple the first binding agent thereto; c) replacing the flowof the first binding agent-containing fluid with a laminar flow of adeactivating fluid, and positioning the interface laterally such thatthe deactivating fluid selectively contacts at least the first bindingagent-coupled area but less than the whole reactive surface area fordeactivation thereof; and d) replacing the flow of the deactivatingfluid with a laminar flow of a fluid containing the second bindingagent, and positioning the interface laterally such that the secondbinding agent-containing fluid selectively contacts the deactivated areaand an adjacent second reactive area of the substrate surface toselectively couple the second binding agent to the second area.
 3. Themethod according to claim 2, which additionally comprises the step of:e) replacing the flow of the second binding agent-containing fluid witha laminar flow of deactivating fluid, and positioning the interfacelaterally such that the deactivating fluid selectively contacts at leastthe deactivated area and the second binding agent-coupled area of thesubstrate surface for deactivation thereof.
 4. The method according toclaim 3, wherein steps b) to e) are repeated at least once to couple atleast one additional binding agent to a respective reactive area on thesubstrate surface.
 5. The method according to claim 1, wherein procedureB comprises the steps of: a) providing a substrate surface, at leastpart of which is reactive to permit coupling of binding agents thereto;b) passing over the substrate surface a laminar flow of a deactivatingfluid, and adjacent thereto a laminar flow of a blocking fluid that doesnot interact with the substrate surface, such that the two fluids flowtogether in the same direction with an interface to each other, andadjusting the relative flow rates of the two laminar fluid flows toposition the interface such that the deactivating fluid selectivelycontacts a first reactive area of the substrate surface for deactivationthereof; c) replacing the flow of the deactivating fluid with a laminarflow of a fluid containing a first binding agent, and positioning theinterface laterally such that the first binding agent-containing fluidselectively contacts the deactivated area and an adjacent secondreactive area of the substrate surface to selectively couple the firstbinding agent to the second area; d) replacing the flow of the firstbinding agent-containing fluid with a laminar flow of activating fluid,and positioning the interface laterally such that the activating fluidselectively contacts at least a part of the deactivated first area foractivation thereof; and e) replacing the flow of the activating fluidwith a laminar flow of a fluid containing a second binding agent, andpositioning the interface laterally such that the second bindingagent-containing fluid selectively contacts the activated first area toselectively couple the second binding agent thereto.
 6. The methodaccording to claim 5, wherein steps b) to e) are repeated at least onceto couple at least one additional binding agent to a respective reactivearea on the substrate surface.
 7. The method according to claim 1,wherein at least one of the reactive areas of the substrate surfacecomprises activated functional groups.
 8. The method according to claim2, wherein step a) comprises passing a laminar flow of activating fluidover at least a part of the substrate surface to provide the at leastpartly reactive surface.
 9. The method according to claim 5, whereinstep a) comprises passing a laminar flow of activating fluid over atleast a part of the substrate surface to provide the at least partlyreactive surface.
 10. The method according to claim 2, wherein step a)comprises providing a surface with pre-activated functional groups. 11.The method according to claim 5, wherein step a) comprises providing asurface with pre-activated functional groups.
 12. The method accordingto claim 1, wherein at least one of the reactive areas of the substratesurface comprises functional groups capable of reacting with the bindingagents without activation of the functional groups.
 13. The methodaccording to claim 2, which comprises the step of, prior to performingstep b), passing over the substrate surface a laminar flow ofdeactivating fluid and an adjacent laminar flow of blocking fluid, andpositioning the interface such that the deactivating fluid selectivelycontacts a reactive edge area of the substrate surface adjacent to thefirst area to be contacted with binding agent in step b) fordeactivation thereof.
 14. The method according to claim 3, whichcomprises performing steps b) to e) in the opposite lateral direction tothe flow path to couple at least one additional binding agent to arespective reactive area of the substrate surface.
 15. The methodaccording to claim 14, which comprises activating an additional part ofthe substrate surface prior to performing steps b) to e) of claim 2 inthe opposite lateral direction to the flow path.
 16. The methodaccording to claim 3, wherein in steps c) and e) the laminar flow ofdeactivating fluid is adjusted to contact also a reactive area adjacentto the binding agent-coupled area to provide a non-coupled area betweenneighbouring binding agent-coupled areas.
 17. The method according toclaim 1, wherein procedure B comprises immobilizing an additionalbinding agent to a third area situated on the other side of the secondarea immobilized with the first binding agent by selectively contactingthe third area with a fluid containing the additional binding agent. 18.The method according to claim 17, wherein the third area is activatedprior to immobilizing the additional binding agent.
 19. The methodaccording to claim 1, which comprises the steps of: a) passing over thesubstrate surface a laminar flow of an activating fluid, and adjacentthereto a laminar flow of a blocking fluid that does not interact withthe substrate surface, and positioning the interface between the twofluids such that the activating fluid selectively contacts a part of thesubstrate surface for activation thereof; b) replacing the flow ofactivating fluid with a laminar flow of blocking fluid, replacing theblocking fluid with a laminar flow of a fluid containing a first bindingagent, and positioning the interface such that the fluid containing thefirst binding agent selectively contacts the non-activated part of thesurface and an adjacent first area of the activated part of the surfaceto couple the first binding agent to the first area; c) replacing theflow of the first binding agent-containing fluid with a laminar flow ofblocking fluid, replacing the blocking fluid with a laminar flow of afluid containing a second binding agent, and positioning the interfacesuch that the binding agent-containing fluid selectively contacts asecond area of the activated part of the surface adjacent to the firstarea coupled with the first binding agent to couple the second bindingagent to the second area; d) replacing the flow of the second bindingagent-containing fluid with a laminar flow of a deactivating fluid, andpositioning the interface such that the deactivating fluid selectivelycontacts the first and second areas coupled with the first and secondbinding agents, respectively; e) replacing the flow of deactivatingfluid with a laminar flow of blocking fluid, replacing the flow ofblocking fluid with a laminar flow of activating fluid, and positioningthe interface such that the activating fluid selectively contacts atleast a portion of the non-activated part of the surface, adjacent tothe part activated in step a), for activation thereof; and f) replacingthe flow of blocking fluid with a laminar flow of a fluid containing athird binding agent, replacing the flow of activating fluid with alaminar flow of blocking fluid, and positioning the interface such thatthe binding agent-containing fluid selectively contacts the first andsecond areas coupled with the first and second binding agents,respectively, and a third area comprising at least a part of the surfaceportion activated in step e) to couple the third binding agent to thethird area.
 20. The method according to claim 19, which additionallycomprises the step of: g) replacing the flow of binding agent-containingfluid with a laminar flow of a deactivating fluid, and positioning theinterface such that the deactivating fluid contacts at least the areacoupled with the third binding agent for deactivation thereof.
 21. Themethod according to claim 19, which comprises coupling a fourth bindingagent to a remaining activated area adjacent to the third area aftercoupling of the third binding agent.
 22. The method according to claim1, which comprises the steps of: a) passing over the substrate surface alaminar flow of a fluid containing an activating fluid, and adjacentthereto a laminar flow of a blocking fluid that does not interact withthe substrate surface, and positioning the interface between the twofluids such that the activating fluid selectively contacts a part of thesubstrate surface for activation thereof; b) replacing the flow ofactivating fluid with a laminar flow of blocking fluid, replacing theblocking fluid with a laminar flow of a fluid containing a first bindingagent, and positioning the interface such that the fluid containing thefirst binding agent selectively contacts the non-activated part of thesurface and an adjacent first area of the activated part of the surfaceto couple the first binding agent to the first area; c) replacing theflow of the first binding agent-containing fluid with a laminar flow ofblocking fluid, replacing the blocking fluid with a laminar flow of afluid containing a second binding agent, and positioning the interfacesuch that the binding agent-containing fluid selectively contacts asecond area of the activated part of the surface adjacent to the firstarea coupled with the first binding agent to couple the second bindingagent to the second area; d) replacing the flow of bindingagent-containing fluid with a laminar flow of blocking fluid, replacingthe flow of blocking fluid with a laminar flow of activating fluid, andpositioning the interface such that the activating fluid selectivelycontacts at least a portion of the non-activated part of the surface,adjacent to the part activated in step a), for activation thereof; ande) replacing the flow of activating fluid with a laminar flow of a fluidcontaining a third binding agent, and positioning the interface suchthat the binding agent-containing fluid selectively contacts the areaactivated in step d) to couple the third binding agent thereto.
 23. Themethod according to claim 22, which additionally comprises the step of:f) replacing the flow of binding agent-containing fluid withdeactivating fluid, and positioning the interface such that thedeactivating fluid contacts at least areas coupled with the bindingagents and previously not deactivated for deactivation thereof.
 24. Themethod according to claim 22, which comprises coupling a fourth bindingagent to a remaining activated area adjacent to the third area aftercoupling of the third binding agent.
 25. The method according to claim22, which comprises deactivating a part of the surface portion activatedin step d), and after coupling the third binding agent in step e),reactivating the deactivated area and coupling a fourth binding agentthereto.
 26. The method according to claim 1, wherein the substratesurface is a sensing surface of a sensor.
 27. The method according toclaim 26, wherein the sensing surface is provided in a flow cell. 28.The method according to claim 27, wherein the flow cell comprises twoinlets and one outlet.
 29. A method of analysing a fluid sample for atleast one analyte, comprising sensitising a sensing surface with atleast two different binding agents according to the method of claim 1,contacting the sensitised areas with the fluid sample, and detectinginteraction between the analyte and the sensing surface.
 30. A method ofanalysis, comprising sensitising a sensing surface with at least twodifferent binding agents according to the method of claim 1, contactingthe sensitised areas with at least one analyte, and studying interactionbetween the analyte and the sensing surface.
 31. The method of claim 1,wherein a system comprising: a flow cell having an inlet end and anoutlet end, at least one sensing surface on a wall surface within theflow cell located between the inlet and outlet ends, wherein the flowcell has at least two inlet openings at the inlet end and at least oneoutlet opening at the outlet end, such that separate laminar flowsentering the flow cell through the respective inlet openings can flowside by side in the same direction through the flow cell over thesensing surface, means for applying laminar flows through the inletopenings, such that the laminar flows pass side by side through the flowcell over the sensing surface with an interface to each other that isparallel to the direction of the laminar flows; and means for varyingthe relative flow rates of the laminar fluids to displace laterally theinterface over the sensing surface; is used for for performing themethod.
 32. A computer program product comprising program code means forperforming the steps of claim 1 when the program is run on a computer.33. A computer program product stored on a computer readable medium,comprising a readable program for causing processing means in orassociated with a system comprising: a flow cell having an inlet end andan outlet end, at least one sensing surface on a wall surface within theflow cell located between the inlet and outlet ends, wherein the flowcell has at least two inlet openings at the inlet end and at least oneoutlet opening at the outlet end, such that separate laminar flowsentering the flow cell through the respective inlet openings can flowside by side in the same direction through the flow cell over thesensing surface, means for applying laminar flows through the inletopenings, such that the laminar flows pass side by side through the flowcell over the sensing surface with an interface to each other that isparallel to the direction of the laminar flows; and means for varyingthe relative flow rates of the laminar fluids to displace laterally theinterface over the sensing surface; to control the execution of thesteps claim 1.